JPWO2016204120A1 - Bamboo fermentation extract production method and immunostimulant - Google Patents

Abstract

To provide new uses for bamboo based on new functions. The method for producing a bamboo fermented extract includes a pulverizing step of pulverizing bamboo to obtain bamboo powder, a fermentation step of fermenting bamboo powder with lactic acid bacteria to obtain a bamboo fermented product, and extracting the bamboo fermented product by hot water extraction And obtaining an extraction step. [Selection] Figure 1

Description

  The present invention relates to a method for producing a bamboo fermented extract obtained by subjecting bamboo fermented material obtained by subjecting bamboo to lactic acid fermentation, an immunostimulant containing the fermented bamboo extract as an active ingredient, and an immunostimulating food composition.

  Bamboo is a grass family plant mainly found in Asia including Japan. Since bamboo grows quickly and is easy to cut, it has been widely used as a material for construction materials, crafts, and daily necessities since ancient times. However, with the advent of chemical materials such as synthetic resins, the use of bamboo has decreased, and there has been a problem that bamboo forests left uncut are eroding fields and farmland. In addition, even in the managed bamboo forest, there was no takeover of the harvested bamboo, and there was a problem that the disposal cost was high.

  Therefore, research and development on new uses of bamboo is underway to make effective use of bamboo. For example, Patent Document 1 describes that the skin portion of bamboo has an antibacterial action, and that bamboo fine powder obtained from the skin portion of bamboo is used as a deodorizing antibacterial material. Patent Documents 2 to 4 by the inventors of the present application describe lactic acid-fermented bamboo powder obtained by lactic acid fermentation of bamboo powder obtained by crushing bamboo material. It describes that it can be applied to plant fertilizers, soil conditioners, deodorant products, antibacterial products and the like.

Japanese Patent No. 2524944 Patent No. 5,357,459 JP 2014-64564 A JP 2014-64565 A

  Various new uses of bamboo as described above have been proposed, but in order to further use bamboo, uses based on new functions that increase the value of bamboo are expected.

  The present invention has been devised in view of the above points, and an object thereof is to provide a new use of bamboo based on a new function.

  Another object of the present invention is to provide a new use of bamboo based on the technology related to lactic acid fermented bamboo powder that the present inventors have proposed so far.

  The inventor of the present application has found that an extract having an immunostimulatory effect can be obtained by subjecting bamboo fermented material obtained by fermenting bamboo with lactic acid bacteria to hot water extraction treatment, and has completed the present invention. Therefore, the method for producing a bamboo fermented extract of the present invention that solves the above problems includes a pulverizing step of pulverizing bamboo to obtain bamboo powder, a fermentation step of fermenting bamboo powder with lactic acid bacteria to obtain a bamboo fermented product, and bamboo An extraction step of extracting the fermented product with hot water to obtain an extract. Thereby, the bamboo fermentation extract which has an immunostimulatory effect can be obtained easily. By using bamboo powder obtained by pulverizing bamboo as a fermentation raw material, the surface area with which the cells come into contact increases, and saccharides, amino acids, vitamins, etc. contained in the cells are leached into the bamboo powder due to pulverization, resulting in a fine honeycomb Since the lactic acid bacteria that lived in the structure-like soft tissue and the like are also dispersed in the bamboo flour, the lactic acid bacteria grow efficiently and the fermentation is promoted. And, since the bamboo fermented extract having an immunostimulatory action can be obtained by extracting the bamboo fermented product with hot water, the safety is high, including food, beverages, cosmetics and medicines, livestock feed, aquatic additives or It can be safely applied to various products such as agricultural preparations.

  Moreover, it is preferable that the manufacturing method of the bamboo fermentation extract of this invention further has a precipitation production | generation process which adds alcohol to an extract or its concentrate, and obtains precipitation. Thereby, the bamboo fermented extract which has the improved immunostimulatory effect is obtained.

  Moreover, it is also preferable to add alcohol so that the final concentration of alcohol may be 60 to 80% by weight in the above-described precipitation generation step. This selects a suitable concentration of alcohol to be added for precipitation formation.

  Furthermore, in the extraction step described above, it is also preferable that hot water extraction of the bamboo fermented product is performed under conditions of 80 ° C. to 130 ° C. and 0.1 to 0.3 MPa. Thereby, suitable temperature and pressure conditions for hot water extraction are selected.

  Moreover, it is also preferable that the bamboo in the manufacturing method of the bamboo fermented extract of this invention is a stem part of a bamboo. By using only bamboo stalks as fermentation materials, the growth of microorganisms becomes active and fermentation is promoted. Moreover, since the water | moisture content adjustment for fermentation becomes easy, a fermentation state is stabilized.

  Moreover, the immunostimulating food composition of the present invention contains the bamboo fermentation extract obtained by the above-mentioned process as an active ingredient. Since the immunostimulatory food composition of the present invention has an action of activating the innate immune function in particular, when it is ingested as a food, the innate immune function, that is, a rapid action against viruses, bacteria, etc. via the intestinal immunity mechanism. It can activate and enhance actions such as effective defense against infections and attacks against malignant tumors.

  Furthermore, the immunostimulant of the present invention is a pulverization step of pulverizing bamboo to obtain bamboo powder, a fermentation step of fermenting bamboo powder with lactic acid bacteria to obtain a bamboo fermented product, hot bamboo extraction of bamboo fermented product and extraction liquid Alcohol is added to the obtained extraction process and the extract or its concentrated liquid, and the bamboo fermentation extract obtained through the precipitation production | generation process which obtains precipitation in order is contained as an active ingredient. The immunostimulant of the present invention particularly has an action of activating the innate immune function. Thereby, it is possible to activate and enhance the natural immunity function of the organism, that is, the action such as rapid infection defense against viruses and bacteria and attack against malignant tumors.

  In the method for producing a bamboo fermentation extract of the present invention, it is also preferable to add a culture containing at least a lactic acid bacterium belonging to the genus Weissella in the above-described fermentation step. Thereby, since the suitable microbial species for obtaining the bamboo fermentation extract of this invention is added as a starter, the time concerning a fermentation process is shortened.

  In addition, the method for producing a bamboo fermentation extract of the present invention comprises a seed preparation step for fermenting bamboo powder with lactic acid bacteria to obtain a bamboo fermented product before the fermentation step, and is obtained in the seed preparation step in the fermentation step. It is also preferable to add the fermented bamboo. Thereby, since the fermentation microorganism acclimatized to the fermentation of bamboo powder is added as a starter, the time required for the fermentation process is shortened.

ADVANTAGE OF THE INVENTION According to this invention, the manufacturing method of a fermented bamboo extract which has the following outstanding effects, an immunostimulant, and an immunostimulatory food composition can be provided.
(1) It has an immunostimulatory action, particularly an action that activates the innate immune function. Moreover, since it has the effect | action which induces the production | generation of IL-12 and IFN-gamma, it has the infection defense effect with respect to a virus or bacteria, the improvement of an immune function fall, an antitumor effect, etc.
(2) Since the raw material used is bamboo and it is extracted using water or alcohol, it is highly safe, food, functional food, beverages, cosmetics, pharmaceuticals, livestock feed, aquatic additives, agricultural preparations, etc. It can be applied to various products with confidence.
(3) Since bamboo is obtained by fermenting lactic acid bacteria and then extracting with hot water, it can be produced relatively easily.
(4) It can contribute to the effective use of bamboo.

It is a flowchart which shows schematically the manufacturing method of the bamboo fermentation extract which concerns on 1st embodiment of this invention. It is a flowchart which shows schematically the manufacturing method of the bamboo fermentation extract which concerns on 2nd embodiment of this invention. It is a graph which shows the immuno-functional activity measurement result of the bamboo fermentation extract of this invention in Example 5. It is a graph which shows the immunofunctional activity measurement result of the lactic acid fermentation bamboo liquid in the comparative example 1. It is a graph which shows the immunofunctional activity measurement result of the bamboo fermentation extract in Example 8. It is a graph which shows the IL-12 production induction result of the bamboo fermentation extract in Example 10. It is a graph which shows the IFN-gamma production induction result of the bamboo fermentation extract in Example 10.

  First, with reference to FIG. 1, the manufacturing method of the bamboo fermented extract which concerns on 1st embodiment of this invention is demonstrated.

  As shown in FIG. 1, the method for producing a bamboo fermented extract P1 according to the present invention includes a step S0 for preparing bamboo as a raw material, a step S1 for crushing bamboo into bamboo powder, and fermenting bamboo powder to bamboo. It is roughly composed of a fermentation step S2 for obtaining a fermented product and a hot water extraction step S3 for extracting the bamboo fermented product with hot water.

(Preparation of bamboo)
First, step S0 for preparing bamboo shown in FIG. 1 will be described. Bamboo, which is a raw material for the bamboo fermented extract according to the present invention, includes a wide variety of gramineous plants generally called bamboo, bamboo grass and bamboo. Specific examples include, but are not limited to, Phyllostachys heterocycla f.pubescence, Phyllostachys bambusoides, Phyllostachys nigra, Tetragonocalamus quadrangularis, Pleioblastus Simonii, Pseudosasa jap Kumazasa (Sasa veitchii) etc. are mentioned, and Miso bamboo is preferably used. Bamboo can be used as it is, so it can be used as it is for the fermented bamboo extract of the present invention without pretreatment other than washing the soil attached to the bamboo. . Raw bamboo is pulverized and powdered or cut into chips, so that the lactic acid bacteria present in the bamboo stems, especially the fine honeycomb-like soft tissue, are expressed and dispersed, and fermentation by lactic acid bacteria derived from bamboo Progresses. Among bamboo, leaf parts such as bamboo leaves contain a lot of antibacterial substances, and there is a high possibility of hindering lactic acid bacteria fermentation in fermentation step S2 to be described later. It is preferable to use only in the following steps.

(Pulverization)
Next, the bamboo crushing step S1 will be described. The method of crushing bamboo may be any method as long as it can be made into powder, and is not particularly limited. For example, bamboo powder having a rotary blade that operates when the cylindrical end surface of raw bamboo comes into contact with it. Can be used. In this type of bamboo powdering apparatus, the raw bamboo is crushed into bamboo powder of a predetermined size by continuously cutting the cylindrical end surface of the raw bamboo in a direction substantially parallel to the cylindrical end surface. By crushing bamboo by scraping the cylindrical end surface of raw bamboo with a rotary blade, a large number of lactic acid bacteria present in the soft tissue and vascular sheath of the stem of the bamboo are exposed, and the fermentation in the subsequent fermentation step S2 Is promoted. Moreover, by crushing bamboo, the cell wall is destroyed, and sugars such as glucose, amino acids and vitamins contained in the cells are dispersed in the powder, and the nutritional requirements of lactic acid bacteria in the fermentation step S2 are satisfied. Fermentation proceeds efficiently.

  The size of the bamboo powder particle is not particularly limited, but if it is too fine, handling becomes difficult, and if it is too large, the particle size is preferably about 50 μm to 1 mm from the viewpoint of decreasing the fermentation rate. 200 μm to 600 μm is more preferable. Moreover, it is also preferable to introduce the bamboo powder obtained by the pulverization process into a metal removing device to adsorb and remove foreign matters due to the spilling of the rotary blade of the bamboo powdering device. Furthermore, the bamboo powder obtained by the pulverization process may be introduced into a sieving apparatus so that the particle size range of the obtained bamboo powder is made uniform, or uncut pieces that have not been pulverized in the pulverization step S1 may be removed.

(fermentation)
Next, fermentation process S2 is demonstrated. Bamboo powder is fermented with lactic acid bacteria derived from bamboo. Specifically, for example, the bamboo powder obtained by the pulverization step S1 described above is packed in a fermentation container, for example, a fermentation bag such as a transparent resin bag, by a predetermined amount, and after removing air inside the fermentation bag as much as possible, the fermentation bag The mouth side of the container is sealed to make the inside of the container substantially anaerobic. This fermentation bag is covered with a light-shielding sheet such as straw and left to stand for about 5 to 20 days in an environment of 20 to 30 ° C., whereby a lactic acid fermentation product of bamboo powder is obtained. The fermentation state can be grasped from the pH value of the bamboo powder. That is, as lactic acid fermentation progresses, lactic acid is produced in the bamboo powder, and the pH exhibited by the bamboo powder becomes acidic. Therefore, after adding 40 g of bamboo powder to 100 mL of purified water and stirring for 5 minutes, it is allowed to stand for 2 minutes. When the pH of the filtrate filtered with the filter paper becomes 4.3 or less, it is determined that the fermentation has sufficiently progressed, and the fermentation step S2 can be finished.

  The fermented bamboo obtained in the fermentation step S2 was analyzed for microflora by denaturant gradient gel electrophoresis (DGGE method). In the initial stage of fermentation, mainly Leuconostoc and Weisella ( Lactic acid bacteria belonging to the genus Weissella were observed, and it was confirmed that lactic acid bacteria belonging to the genus Weissella were the main component of the microbiota in the fermented bamboo ferment. From this, it is also preferable to use a culture containing lactic acid bacteria belonging to the genus Weissella as a starter in the fermentation step S2. As a result, the dominant species of the microflora in the bamboo flour can be promptly promoted as a genus of Weissella, and the fermentation days in the fermentation step S2 can be shortened by about 30%, which is about 3 to 15 days. be able to. In addition, the microbial flora and the number of microorganisms vary depending on temperature fluctuations, water content and type of bamboo, etc. By using a starter, a stable bamboo ferment can be obtained throughout the year. The starter is preferably added with a starter containing Weissella lactic acid bacteria separately cultured in a liquid medium so as to be 0.5 mL to 5 mL per 1 kg of bamboo powder.

  Moreover, it is also preferable to use as a starter the fermented bamboo obtained by fermenting bamboo powder beforehand in fermentation process S2. In this way, by providing an inoculum preparation step in advance, and using the obtained bamboo fermented product as a starter, the dominant species Weissella contained in the bamboo fermented product is a new one that has just undergone the grinding step S1. Fermentation of bamboo powder can be promoted, the fermentation days in the fermentation step S2 can be shortened by about 30%, and can be about 3 to 15 days. Moreover, by using a starter, a stable bamboo fermented product can be obtained regardless of the climate and the state of the bamboo. Thus, when using the bamboo fermented material prepared beforehand as a starter, it is preferable to add so that it may become 5-20g per 1 kg of new bamboo flour. Furthermore, it is also effective to use a part of the fermented bamboo obtained in the main fermentation step S2 as a starter for the next fermentation step.

  Moreover, it is also preferable to perform fermentation in fermentation process S2 on low vacuum or medium vacuum conditions, or inert gas atmosphere like nitrogen gas. As a result, the growth of yeast such as Hansenula or Pichia in bamboo flour is prevented, and the generation of ethyl acetate generated from acetic acid and ethanol produced by the yeast can be prevented.

(Hot water extraction)
Next, hot water extraction process S3 is demonstrated. In this hot water extraction step S3, the bamboo fermented product obtained in the fermentation step S2 is extracted with hot water to obtain a bamboo fermented extract P1. Examples of the hot water extraction method include reflux hot extraction and pressurized hot water extraction using an autoclave apparatus. Specifically, for example, a bamboo fermented product and water are placed in a reaction vessel, and a refluxed heat treatment or the like is performed for a predetermined time to extract the bamboo fermented product extract. Water is preferably added in an amount of 4 to 15 liters, more preferably 4 to 10 liters, per 1 kg of bamboo fermented product. As the extraction solvent, water is used, but it is possible to contain other components as long as the extraction efficiency is not hindered and the action of the resulting bamboo fermentation extract is not affected. As extraction temperature, it is preferable to set it as 80 degreeC or more, 80 to 130 degreeC is more preferable, and 100 to 121 degreeC is especially preferable. Extraction is performed under atmospheric pressure or under pressure, and the pressurizing condition is preferably 0.1 MPa to 0.5 MPa, more preferably 0.1 MPa to 0.3 MPa. The extraction time is preferably 10 minutes to 5 hours, more preferably 20 minutes to 2 hours. After the hot water extraction treatment, the bamboo fermentation extract P1 is obtained by removing the residue. As the bamboo fermentation extract P1 of the present invention, in addition to the bamboo fermentation extract P1 itself obtained in the hot water extraction step S3, the bamboo fermentation extract P1 is made into a concentrated liquid by concentration treatment such as vacuum concentration, bamboo fermentation The extract P1 is subjected to a drying treatment such as a freeze-drying treatment to form a solid or powder.

  Next, with reference to FIG. 2, the manufacturing method of the bamboo fermented extract which concerns on 2nd embodiment of this invention is demonstrated.

  As shown in FIG. 2, the method for producing the bamboo fermented extract P2 of the present invention includes a step S0 for preparing bamboo as a raw material, a crushing step S1 for crushing bamboo into bamboo powder, and fermenting bamboo powder to bamboo. A fermentation process S2 for obtaining a fermented product, an extraction process S3 for extracting the bamboo fermented product with hot water, and a precipitation generating process S4 for obtaining a precipitate from the hot water extract are roughly constituted. Among these steps S0 to S4, the bamboo preparation step S0, the pulverization step S1, the fermentation step S2 and the hot water extraction step S3 are the same as the steps S0 to S3 in the first embodiment described above, respectively. Since the effect is also the same, description is abbreviate | omitted.

(Precipitation formation)
The precipitation generation step S4 will be described. In this precipitation production | generation process S4, alcohol is added to the extract obtained by hot water extraction process S3, precipitation is produced | generated, and bamboo fermentation extract P2 is obtained by collect | recovering the precipitation. Examples of the alcohol used include ethanol and isopropanol. Ethanol is preferable from the viewpoint of excellent safety. The alcohol may be added in such an amount and concentration that an insoluble precipitate is formed in the extract. From the viewpoint of efficiently promoting the formation of a precipitate, specifically, the alcohol in the mixed solution composed of the extract and alcohol is used. It is preferably added so that the final concentration of alcohol is 50% by weight to 85% by weight, and is preferably added so that the final concentration of alcohol is 60% by weight to 80% by weight, It is particularly preferable to add 75% by weight. In addition, from the viewpoint of reducing the amount of alcohol added to the extract, it is also preferable to subject the extract before addition of alcohol to a vacuum concentration treatment or the like to prepare a concentrate. In this case, the concentration ratio of the extract varies depending on the amount of water added to the bamboo fermented product in the hot water extraction step S3, but it is preferably 3 to 20 times concentrated, and 5 to 15 times concentrated. Is more preferable. By adding a predetermined amount of alcohol to the hot water extract or its concentrated solution, components having an immunostimulatory effect contained in the extract are selectively precipitated, and unnecessary components are removed and purified bamboo fermentation. Extract P2 is obtained. The obtained precipitate, that is, the bamboo fermentation extract P2, is recovered by solid-liquid separation by filtration, centrifugation, or the like. The bamboo fermentation extract P2 is then dried and stored as a solid or powder.

  The bamboo fermentation extract of the present invention has an immunostimulatory action. The immunostimulatory effect referred to in the present invention means an effect of improving the functions of both innate immunity and acquired immunity, but the immunostimulant containing the bamboo fermentation extract according to the present invention as an active ingredient is particularly innate immunity. It has an effect of activating the function, and has an effect of inducing IL-12 and IFN-γ production. Thereby, it is possible to activate and enhance the innate immune function, that is, actions such as rapid infection protection against pathogens and attacks against malignant tumors. In addition, the immune function is activated when the immune function is reduced due to causes such as aging, stress, and fatigue, or the immune function is stably maintained even when exposed to causes such as aging, stress, and fatigue. It also has the effect of Moreover, since the bamboo fermentation extract of the present invention has an IL-12 and IFN-γ production-inducing action, it not only activates Th1 cells to enhance cellular immunity but also promotes cellular immunity. Due to the enhancement, it is also expected to have an action of suppressing humoral immunity and suppressing allergic reaction.

  The immunostimulant of the present invention can be used in various products such as food, beverages, cosmetics, quasi-drugs, and pharmaceuticals, as well as livestock feed, aquatic additives or agricultural preparations. Bamboo fermented extract, which is an active ingredient, has discovered a new function of bamboo that has been used for food such as bamboo shoots, is highly safe, and can be taken on a daily basis. Therefore, it is preferably used as a food or beverage composition. The immunostimulating food composition containing this bamboo fermented extract as an active ingredient is supplement forms such as tablets, capsules, granules, syrups, beverages, confectionery, bread, rice cakes, cereals, noodles, jelly, soup, It can be used in all forms such as dairy products, seasonings and edible oils. Moreover, when using as a food composition, it is also possible to combine various active ingredients, nutrients such as vitamins, minerals or amino acids in various ways within a range not affecting the efficacy of the active ingredients of the present invention. Foods developed from the food composition of the present invention include supplements, health foods, functional foods, foods for specified health use, and the like. It can also be used for animals other than humans (pets, livestock, aquaculture animals, etc.), and in that case, it should be used in the form of feed, pet food, animal supplements or animal beverages, etc. Can do.

  When using the immunostimulant of this invention as a pharmaceutical or a quasi-drug, it can be prepared into various forms by a conventionally used method. In this case, pharmacologically acceptable carriers and excipients for normal formulations, lubricants, dispersants, disintegrants, buffers, solvents, extenders, preservatives, fragrances, stabilizers, etc. It can be formulated using an additive that is acceptable as an additive. Furthermore, in order to improve bioavailability and stability, a drug delivery system including a preparation technique such as microcapsule, liposome preparation, fine powder or inclusion using cyclodextrin or the like can also be used. Examples of the dosage form include tablets, granules, capsules, syrups and the like, but are not particularly limited thereto.

  The pharmaceutical or food composition containing the immunostimulant of the present invention is preferably 1 mg to 10 g, more preferably 1 mg to 1 g in terms of dry weight, as a daily intake. preferable.

  Examples of the present invention will be described below, but the present invention is not limited to these examples.

[Example 1]
1. Manufacture of Bamboo Fermentation Extract (1) Munetake bamboo with a stem height of about 4 m was cut down, the leaves were cut off with straw to make only the stems, and then washed with water to remove mud and dirt. About 4 m of the stem part of this Somune bamboo was pulverized using a bamboo powdering device (manufactured by Marudai Iron Works Co., Ltd., model number: PA-Z) to obtain about 10 kg of bamboo powder of 500 μm or less. Next, 10 kg of ground bamboo powder was put into a 15 kg capacity resin bag, the outer periphery of the resin bag was pressed to remove as much air inside the bag as possible, and the bag was sealed. The resin bag was covered with a light shielding sheet and allowed to stand for 14 days in a 25 ° C. environment. A portion of the fermented bamboo after 14 days was taken and microbiota analysis was performed by denaturing gradient gel electrophoresis (DGGE method). It was confirmed that 1 kg of the obtained bamboo fermented product was put in a reaction vessel for reflux heating, 5 L of water was poured, and hot water extraction was performed by heating to reflux. The reaction time was 60 minutes. After completion of the reaction, the mixture in the reaction vessel was filtered, and the filtrate (about 4.5 L) was recovered as an extract. To 4.5 L of this extract, 6 L of a 15% aqueous ethanol solution was added to precipitate insoluble components, and the precipitate was collected. The collected precipitate was freeze-dried to obtain about 30 g of bamboo fermentation extract (1).

[Example 2]
2. Manufacture of Bamboo Fermentation Extract (2) A bamboo shoot with a stem height of about 4 m was cut down, the leaves were cut off with a cocoon to make only the stem, and then washed with water to remove mud and dirt. About 4 m of the stem part of this Somune bamboo was pulverized using a bamboo powdering device (manufactured by Marudai Iron Works Co., Ltd., model number: PA-Z) to obtain about 10 kg of bamboo powder of 500 μm or less. 100 g of the bamboo fermented product obtained in Example 1 was added to 10 kg of bamboo powder and mixed well. Next, this bamboo powder was put into a 15 kg capacity resin bag, the outer periphery of the resin bag was pressed to remove as much air as possible from the inside of the bag, and the bag was sealed. The resin bag was covered with a light-shielding sheet and allowed to stand in a 25 ° C. environment for 9 days. 1 kg of the obtained bamboo fermented product was put in a reaction vessel for reflux heating, 5 L of water was poured, and hot water extraction was performed by heating to reflux. The reaction time was 60 minutes. After completion of the reaction, the mixture in the reaction vessel was filtered, and the filtrate (about 4.5 L) was recovered as an extract. To 4.5 L of this extract, 6 L of a 15% aqueous ethanol solution was added to precipitate insoluble components, and the precipitate was collected. The collected precipitate was freeze-dried to obtain about 30 g of bamboo fermentation extract (2).

[Example 3]
3. Manufacture of bamboo fermented extract (3) for immune functional activity test The bamboo fermented product obtained in Example 1 was sampled in sterilized physiological saline (0.9% NaCl) to a sample concentration of 100 mg / mL. Suspended. This suspension was subjected to hot water extraction treatment of the fermented bamboo product at 121 ° C. for 20 minutes in an autoclave. The hot water extract was centrifuged and the supernatant was collected to obtain a bamboo fermentation extract (3). This supernatant was used for the immunostimulation test. In addition, 1 mL of the collected supernatant was dried using a centrifugal evaporator, and the concentration of the bamboo fermentation extract (3) in the supernatant was calculated from the obtained dry weight.

[Example 4]
4). Production of Bamboo Fermented Extract (4) for Immune Functional Activity Test The bamboo fermented product obtained in Example 2 is sampled in sterile physiological saline (0.9% NaCl) to a sample concentration of 100 mg / mL. Suspended. This suspension was subjected to hot water extraction treatment of the fermented bamboo product at 121 ° C. for 20 minutes in an autoclave. The hot water extract was centrifuged and the supernatant was collected to obtain a bamboo fermentation extract (4). This supernatant was used for the immunostimulation test. In addition, 1 mL of the collected supernatant was dried using a centrifugal evaporator, and the concentration of the bamboo fermentation extract (4) in the supernatant was calculated from the obtained dry weight.

[Example 5]
5. Immune Functional Activity Test As an immune functional activity test, an innate immunity promoting activity test was performed using silkworm muscle contraction as an index. That is, in this test method, when a test sample is administered to a larva of a completely transformed insect such as a silkworm, if a neurotransmitter is present in the test sample, rapid muscle contraction occurs, but the innate immune function activation action This is based on the finding that when a substance having a lysate is administered to a fully transformed insect larva, a slow muscle contraction (gradual muscle contraction) that takes about 10 minutes to complete is produced. The following tests were conducted using the bamboo fermentation extract (3) obtained in Example 3 and the bamboo fermentation extract (4) obtained in Example 4 as test samples.

  The slow muscle contraction of silkworms was measured as follows (see Hamamoto H., Kamimura M., Sekimizu K., J. Biol. Chem., 2008, 283 (4), pp.2185-91. ). A test sample of 50 μL was injected into a decapitation muscle specimen of a 5-day-old silkworm, and the body length of the silkworm was measured. Muscle contraction value (Contraction Value, C value = ([length before contraction (before injection)] − The maximum value (after about 10 minutes) of [length after contraction (after injection)] / [length before contraction]) was determined. The test sample was serially diluted to a predetermined concentration with 0.9% physiological saline as shown in Table 1 below, and the C value was measured for each diluted solution. The slow muscle contractile activity when C value = 0.15 is defined as 1 unit, the amount of the test sample giving C value = 0.15 is obtained from the obtained dose response curve, and the activity per 1 mg of the test sample Was calculated. Further, 50 μL of physiological saline was used as a negative control, and 0.2 mL of air was used as a positive control. The results are shown in Table 1 and FIG. Fig.3 (a) has shown the result of the bamboo fermentation extract (3), FIG.3 (b) has shown the result of the bamboo fermentation extract (4).

[Comparative Example 1]
6). Production of Lactic Acid Fermented Bamboo Fluid for Immune Functional Activity Test and Immune Functional Activity Test 1 kg of bamboo fermented product obtained in Example 1 is put into a 10 L plastic container, and 20 g of bamboo charcoal powder and 6 g of papain enzyme are added and mixed well. did. Further, 8 L of water was poured to close the lid, and the mixture was allowed to stand in a 25 ° C. environment for 14 days. After 14 days, the mixture in the container was filtered, and the filtrate (about 8 L) was recovered as lactic acid fermentation bamboo liquid. 10 mL of this lactic acid fermented bamboo liquid was taken, pH was checked with pH test paper, and neutralized with NaOH. Moreover, 1 mL of lactic-acid-fermented bamboo liquid was made to dry with a centrifugal evaporator, and the density | concentration of the component contained in the lactic-acid-fermented bamboo liquid was computed from the obtained dry weight. The same immunity functional activity test as Example 5 was done by using this lactic acid fermented bamboo liquid as a test sample. The results are shown in Table 2 and FIG.

  As shown in Table 1, the concentration of the bamboo fermentation extract (3) was 15 mg / mL, and the concentration of the bamboo fermentation extract (4) was 11 mg / mL. Moreover, when the immune function activity of these extracts was measured, the dose response curve as shown in FIG. 3 was seen, and it was found that the bamboo fermentation extract of the present invention has innate immunity promoting activity. The specific activity of the bamboo fermentation extract (3) was 2.1 units / mg, and the specific activity of the bamboo fermentation extract (4) was 2.7 units / mg. On the other hand, as for the lactic acid fermented bamboo liquid shown in the comparative example, no dose response curve was observed as shown in FIG. 4, and no innate immunity promoting activity was observed. From these results, it became clear that the fermented bamboo extract obtained by hot water extraction of the fermented bamboo product has an immunostimulatory effect.

[Example 6]
7). Production of Bamboo Fermentation Extract (5) 10 g of the bamboo fermentation product obtained in Example 1 was suspended in 100 mL of sterilized physiological saline (0.9% NaCl). This suspension was subjected to hot water extraction treatment of the bamboo fermented product in an autoclave at 121 ° C. and 2 atm for 20 minutes. The hot water extract was centrifuged to collect the supernatant and dried on a centrifugal evaporator to obtain 440 mg of powdered bamboo fermentation extract (5). Moreover, when the quantity of the saccharide contained in the obtained bamboo fermentation extract (5) was quantified by the phenol sulfuric acid method, it turned out that 7 weight% of the bamboo fermentation extract (5) is a saccharide ( (See Table 4).

[Example 7]
8). Production of Bamboo Fermentation Extract (6) 10 g of the bamboo fermentation product obtained in Example 1 was suspended in 100 mL of sterilized physiological saline (0.9% NaCl). This suspension was subjected to hot water extraction treatment of the bamboo fermented product in an autoclave at 121 ° C. and 2 atm for 20 minutes. The hot water extract was centrifuged and the supernatant was collected. 95% ethanol was added to the collected supernatant, and ethanol was added until the final concentration of ethanol was 68%. The produced precipitate was collected and dried to obtain 27 mg of powdered bamboo fermentation extract (6). Moreover, when the quantity of the saccharide contained in the obtained bamboo fermentation extract (6) was quantified by the phenol sulfuric acid method, it turned out that 52 weight% of the bamboo fermentation extract (6) is a saccharide ( (See Table 4).

[Example 8]
9. Immune functional activity test for bamboo fermentation extract (5) and (6) Conducted using bamboo fermentation extract (5) and bamboo fermentation extract (6) obtained in Example 7 and Example 8 as test samples An immune function activity test was conducted in the same manner as in Example 5. The results are shown in Table 3 and FIG. FIG. 5 (a) shows the result of the bamboo fermentation extract (5), and FIG. 5 (b) shows the result of the bamboo fermentation extract (6).

  When the immune functional activity of the bamboo fermentation extract obtained in Example 6 and Example 7 was measured, the dose response curve shown in FIG. 5 was observed, and the bamboo fermentation extract of the present invention has innate immunity promoting activity. I understood it. Specific activity values determined from this dose response curve were 7.3 units / mg for bamboo fermentation extract (5) and 230 units / mg for bamboo fermentation extract (6) that had undergone the ethanol precipitation formation step ( (See Table 4). From this, the bamboo fermented extract obtained by hot water extraction of the bamboo fermented product has an immunostimulatory action, and the components having the immunostimulatory action are concentrated or selectively obtained by ethanol precipitation. It was clarified that the bamboo fermented extract (6) that has undergone the precipitation generation step exhibits a higher immunostimulatory action. Moreover, as shown in Table 4 below, the bamboo fermentation extract (6) has a saccharide content of 52% by weight. Thus, since the specific activity also increases with the increase in the saccharide content, it is considered that the polysaccharide contained in the bamboo fermentation extract is one of the active ingredients having an immunostimulatory action.

[Example 9]
10. Production of Bamboo Fermentation Extract (7) 1 kg of the bamboo fermentation product obtained in Example 1 was placed in a reaction vessel for reflux heating, poured with 10 L of water, and subjected to hot water extraction at 100 ° C. by heating to reflux. The reaction time was 120 minutes. After completion of the reaction, the mixture in the reaction vessel was filtered, and about 9 L of the filtrate was recovered. The filtrate was concentrated about 10 times to obtain a concentrate. To this concentrate, 95% ethanol was added, and ethanol was added until the final concentration of ethanol was 70%. The precipitate formed by filtering this mixed solution was collected and dried to obtain a powdered bamboo fermentation extract (7).

[Example 10]
11. Immune Response Test Using Mouse Spleen Cells Spleen cells were collected from C57B6 mice and adjusted so that spleen cells were present at 5 × 10 ⁶ cells / mL in RPMI-1640 liquid medium. The bamboo fermentation extract (7) produced in Example 9 was used as the test sample. Moreover, since LPS is often included in natural product-derived extracts, lipopolysaccharide (LPS) is used as a comparative sample, and cytokine production by the bamboo fermentation extract of the present invention is due to LPS. It was confirmed whether or not. Bamboo fermentation extract (7) was added to spleen cells (5 × 10 ⁶ cells / mL) of C57B6 mice at final concentrations of 2 μg / mL, 5 μg / mL and 10 μg / mL, and cultured for 48 hours. Similarly, LPS was added to spleen cells (5 × 10 ⁶ cells / mL) of C57B6 mice to a final concentration of 0.1 μg / mL and cultured for 48 hours. After culturing for 48 hours, the production amounts of IL-12 (p70) and IFN-γ in the medium were measured by ELISA. FIG. 6 shows the results of IL-12 (p70) production, and FIG. 7 shows the results of IFN-γ production. As shown in FIGS. 6 and 7, the bamboo fermentation extract of the present invention was found to induce the production of IL-12 and IFN-γ. In addition, since neither IL-12 nor IFN-γ was observed from spleen cells to which LPS was added, the production of these cytokines by the bamboo fermentation extract of the present invention was not caused by LPS. It was considered that the action was due to components other than LPS contained in the fermented extract.

  The present invention is not limited to the above-described embodiments or examples, and various design changes within the scope not departing from the gist of the invention described in the claims are also included in the technical scope. .

  The bamboo fermented extract of the present invention can be used in the fields of livestock and aquaculture in addition to being used as foods, beverages and pharmaceuticals.

Claims (8)

Crushing process of crushing bamboo to obtain bamboo powder;
Fermenting the bamboo powder with lactic acid bacteria to obtain a bamboo fermented product,
An extraction step of extracting the bamboo fermented product with hot water to obtain an extract, and a method for producing the bamboo fermented extract.   Furthermore, it has a precipitation production | generation process which adds alcohol to the said extract or its concentrate, and obtains precipitation, The manufacturing method of the bamboo fermentation extract of Claim 1 characterized by the above-mentioned.   The method for producing a bamboo fermented extract according to claim 2, wherein the alcohol is added so that a final concentration of the alcohol is 60 to 80% by weight in the precipitation generation step.   The hot-water extraction in the said extraction process is performed on 80 to 130 degreeC and 0.1MPa-0.3MPa conditions, The bamboo fermentation extract of any one of Claims 1-3 characterized by the above-mentioned. Production method.   The method for producing a bamboo fermented extract according to any one of claims 1 to 4, wherein the bamboo is a bamboo stem.   A pulverization step of pulverizing bamboo to obtain bamboo powder, a fermentation step of fermenting the bamboo powder with lactic acid bacteria to obtain a bamboo fermentation product, and an extraction step of extracting the bamboo fermentation product with hot water to obtain an extract in order An immunostimulating food composition containing, as an active ingredient, a bamboo fermentation extract obtained through the above process.   A crushing step of crushing bamboo to obtain bamboo powder, a fermentation step of fermenting the bamboo powder with lactic acid bacteria to obtain a bamboo fermented product, an extraction step of extracting the bamboo fermented product with hot water to obtain an extract, An immunostimulatory food composition comprising, as an active ingredient, a bamboo fermentation extract obtained by sequentially adding an alcohol to an extract or a concentrated solution thereof and a precipitation generating step for obtaining a precipitate. A crushing step of crushing bamboo to obtain bamboo powder, a fermentation step of fermenting the bamboo powder with lactic acid bacteria to obtain a bamboo fermented product, an extraction step of extracting the bamboo fermented product with hot water to obtain an extract, An immunostimulant containing, as an active ingredient, a bamboo fermentation extract obtained by sequentially adding an alcohol to an extract or a concentrated solution thereof and a precipitation generating step for obtaining a precipitate.

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